Excess activation of TNF superfamily (TNFsf) receptors can induce a myriad of pathological conditions (1, 2). While some biological agents have positively impacted the course of these diseases, each carries substantial complications (3). The TNFsf member RANKL is a cytokine that regulates osteoclast formation and function (4-7) and excess activation of its receptor, RANK, can promote many if not most forms of pathological bone loss. RANKL exists as a homotrimer in solution (8). Each of the three interfaces separating the monomers contains a binding groove accepting a single copy of RANK or the anti-osteoclastogenic decoy receptor, osteoprotegerin (OPG) (9-11). As each trimer assembles, loops and strands at the edges of apposed monomers combine to form the sides of the receptor-binding clefts. It is the shape of the binding clefts which determine receptor selectivity. For wild-type RANKL, each of the three identical receptor-binding clefts, spaced equally around the outside of the cytokine, can accept a single copy of RANK or the anti-osteoclastogenic decoy receptor, osteoprotegerin (OPG)
RANKL, a member of the TNF superfamily, binds to multiple receptors (RANK and OPG) with different biological effects. Within this superfamily, there are several examples of cytokines demonstrating receptor promiscuity (17). For example, TNFα, which recognizes TNFR1 and TNFR2, is central to the pathogenesis of disabling disorders such as rheumatoid arthritis and psoriasis (18). Treatment of these diseases has been greatly facilitated by global TNFα blockade using humanized antibodies or soluble receptor (19, 20). As effective as these drugs are, they carry major complications such as predisposition to malignancy and serious infections, including tuberculosis (21, 22). Current evidence indicates that the positive effects of anti-TNFα therapy reflects suppressed activation of TNFR1, while negative consequences are due to inhibition of the pro-immune properties of TNFR2 (23, 24). For example, the osteolysis responsible for orthopedic implant loosening appears to be solely mediated by TNFR1 (Merkel et al., 1999; Am. Journ. Pathol. 154(1):203-210). In fact activation of TNFR1 promotes osteoclastogenesis, leading to bond resorption, while activation of TNFR2 inhibits it. Further, TNFR1 activation inhibits pre-osteoblast differentiation, and so blunts bone formation, while activation of TNFR2 does not (Abbas et al., 2003; Cytokine 22(1-2): 33-41. Thus TNFR2 signaling inhibits bone remodeling, but not bone formation, and so displays protective properties in the context of inflammatory osteolysis. Nago et al, 2011, J. Bone Miner. Metab; 29(6): 671-681. and Hussain et al., 2008, J. Bone Miner. Metab; 26(5):p 469-477). The need for DR4 vs. DR5 specific TRAIL variants stems from the apparent DR4- or DR5-specific sensitivities of various tumors to selective receptor agonists. For example, acute myeloid leukemia appears to be more sensitive to a DR4-selective variant yet resistant to killing by a DR5-selective TRAIL variant (Szegezdi, Journal of Cellular and Molecular Medicine Volume 15, Issue 10, pages 2216-2231, October 2011). Additionally, it has been reported using several different chronic lymphocytic leukemia cell lines that an agonist antibody specific for DR4 induce apoptosis whereas an agonist antibody specific for DR5 fails to do so (Xiao, Leukemia and Lymphoma July 2011, Vol. 52, No. 7, Pages 1290-1301). Conversely, DR5-selective TRAIL variant was effective in killing a breast cancer cell line whereas a DR4-selective TRAIL variant was not despite similar expression of DR4 and DR5 in these cells (Kelley 2005 The Journal of Biological Chemistry, 280, 2205-2212). The sensitivity of various cancer types including primary cells and cell lines is reviewed extensively in van Roosmalen et al. (van Roosmalen Biochemical Pharmacology, Volume 91, Issue 4, 15 Oct. 2014, Pages 447-456). RANKL, TNF, and TRAIL interact with their receptors in a homologous fashion (9, 10, 25, 26).
“Novel variants of RANKL protein” WO2003059281 (PCT/US2003/000393) of Desjarlais, J. R., et al., and TNF Family Ligand Variants” US20140096274 A1 of Quax, W. J., et al. discuss RANKL variants. “Single-chain antagonist polypeptides” WO2001025277 of Andersen, K. V., et al. discusses modification of osteoprotegerin ligand. The articles “Crystal structure of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand specificity” of Lam, J., Nelson, C. A., Ross, F. P., Teitelbaum, S. L., Fremont, D. H; J Clin Invest. 2001 October; 108(7):971-9 and “Structural and functional insights of RANKL-RANK interaction and signaling” of Liu, C., et al., J Immunol. 2010 Jun. 15; 184(12):6910-9 discuss structural features of RANKL.
The sole medically approved inhibitor of this pathway, denosumab, targets the cytokine itself but not its receptor and its effects last for 7-9 months (12). Given the profound suppression of bone remodeling accompanying cytokine removal or other anti-bone resorptive strategies, shorter acting agents are needed.